Diabetes mellitus is a disease of the pancreas which exhibits an increased level of blood sugar as the essential symptom. In most cases, it is not only the beta-islet cells that are affected, but the entire pancreas, so that one might expect there to be a deficiency of all of the pancreatic enzymes, including lipases. It is manifested by an insufficient amount of the pancreatic hormone insulin being released.
At present, the natural hormone is, as a rule, replaced by animal insulin isolated from the glands of slaughtered animals, or human insulin, which is accessible semisynthetically from porcine insulin or by genetic engineering methods.
Two fundamentally different ways have hitherto been taken in the use of genetic engineering methods: separate synthesis of A and B chains and their subsequent chemical recombination, and synthesis of preproinsulin, the naturally occurring precursor of insulin. In the proinsulin molecule, the A and B chains are linked by a connecting piece, the C peptide. According to current theory, the most important function of this piece is spatial fixing of the two chains relative to one another, so that correct folding can take place. When folding has taken place, the three disulfide bridges are linked, and the unmodified three-dimensional structure of the insulin is thus stabilized. The C peptide is split off by enzymes having a tryptic and carboxypeptidase B activity. The splitting sites are predetermined by a Lys-Arg sequence (before the N-terminus of the A chain) or and Arg-Arg sequence (at the C-terminus of the B chain). Only free insulin has full biological activity, because part of the molecule is probably masked in the presence of the C peptide.
The particular chemical nature of insulin means that therapy is as a rule parenteral; the hormone would be completely degraded even before it was able to act, for example, on passage through the stomach and intestine. However, degradation reactions, essentially by various, relatively non-specific proteolytic enzymes, also take place at the injection site and in the circulation. The short in vivo half life of only about 7 minutes which thereby results is in principle appropriate from the physiological point of view in the context of homeostasis; however, therapy is thereby made considerably more difficult, because the diabetic must typically inject himself four times daily, as a rule shortly before mealtimes.
Early attempts have accordingly already been made to impart a protracted action to the insulin. The most successful so far have been those methods in which the insulin is converted into a paringly soluble state by addition of a depot auxiliary. Depot auxiliaries include, above all, divalent zinc ions, in the presence of which the insulin can be in crystalline or amorphous form in a neutral medium. The addition of basic proteins, for example, protamine sulfate or human globin, has the same effect, since insulin is an acid molecule with an isoelectric point p.sub.I of 5.4: basic protein and insulin are in the form of a crystalline or amorphous salt-like, sparingly soluble complex in the neutral range.
It is imagined that the slow release of the insulin from these sustained release formulations takes place by dilution, i.e., diffusion, of individual components which build up the crystal or the amorphous precipitate, or, in the case of insulin complexes with basic proteins, by proteolytic degradation of the depot excipient.
Human proinsulin, either by itself or in combination with the customary depot additions, has recently also been discussed as a delayed action principle, cf. German Patent No. A 3,232,036. The theory is that the proteolytic splitting of the C peptide is delayed in vivo and hence the fully active hormone is released from the proinsulin, which has only little inherent biological activity (about 1/8 of the activity of insulin, based on the amount of protein). Only those proinsulins which are identical or very similar in their sequence to that from humans are acceptable for use in humans. As is generally known, porcine and bovine proinsulin are immunogenic. The exact mode of action of proinsulin, however, is at present still open. It has in no way been proven that insulin is specifically released. On the contrary, degradation in vivo will take place in several ways, with production of in most cases inactive fragments. The therapeutic use of pro-insulin could thus rather be found, if at all, at the receptor level.
Diabetes therapy is characterized by individual influence factors, such as differences in the characteristics of the subcutaneous tissue, and also specific eating habits, physical activities, and many others besides. It is thus absolutely essential for good adjustment of the blood sugar to have available a number of insulin products with different action characteristics which are adapted to the individual requirements. In connection with non-optimum adjustment, in particular the topic of delayed diabetic damage is an issue. The immediate objective and subjective effects include hyper- or hypoglycemia, and macro-and micro-angiopathy, neuropathy, bephropathy, and retinopathy.
Besides pure delayed action insulin, so-called intermediate acting insulins have above all proven to be preparations which are optimally suited to the requirements of the patient. These are mixtures of a delayed action component and a component having an immediate and short action. Such mixtures are in general complicated multiphase systems which remain stable over a long period only when mixed in relatively narrowly defined proportions. Thus, for example, a suspension of 2-zinc-insulin crystals from pigs is not freely miscible with dissolved porcine insulin. The admixed, dissolved insulin precipitates immediately or in the course of time because of the relatively high zinc content which is necessary to stabilize the crystals. Such mixtures are stable within narrow limits if bovine insulin or a mixture of dissolved porcine insulin and phenylalanine (B1)-porcine insulin is used as the dissolved insulin, as disclosed in German patents No. A 2,418,218 and A,2,459,515. From the point of view of miscibility with dissolved insulin, protamine-insulin formulations are more advantageous, if crystals of protamine and insulin are used in an isophane ratio as the delayed action components.
Despite the early discovery of insulin and the later discovery and use of sulfonylureas (e.g., chlorpropamide, tolbutamide, acetohexamide, tolazamide, and biguanides such as phenformin) as oral hypoglycemic agents, the treatment of diabetes is less than satisfactory.
Because the use of insulin for treating diabetes requires multiple daily dosages, it is necessary to estimate frequently the amount of sugar in the urine or in the blood. The administration of an excessive dose of insulin causes hypoglycemia, with effects ranging from mild abnormalities in blood glucose to coma, or even death. Where effective, synthetic hypoglycemic agents are preferred over insulin, since they are more convenient to administer and are less prone to cause severe hypoglycemic reactions. However, the clinically available hypolglycemics are fraught with other toxic manifestations which limit their use. In any event, where one of these agents may fail in an individual case, another may succeed. The need for additional hypoglycemic agents, which may be less toxic or succeed where others fail, is clearly evident.
One attempt to provide non-insulin treatments for diabetes is disclosed by Holland, in U.S. Pat. No. 4,511,575. Holland discloses that certain pyrrolecarboxylic and pyrroleacetic acid derivatives substituted on the pyrrole ring with thioether groups, acyl groups, phenyl, substituted phenyl, phenoxy, substituted phenoxy, benzyl, or halo and optionally substituted on the pyrrole nitrogen with alkyl, and the pharmaceutically acceptable salts thereof can be used to lower the blood glucose levels of hyperglycemic animals.
Iwamura et al., in U.S. Pat. No. 4,472,432, disclose the use of alpha, beta-unsaturated higher fatty acids of the formula EQU CH.sub.3 (CH.sub.2).sub.n CH.dbd.CHCOOH
wherein n represents 10, 12, 14, or 16, and pharmaceutically acceptable salts thereof. These fatty acids, which are extracted from freshwater clams, are said to be effective treatments for diabetes.
Since diabetics are thought to have more agreeable platelets with a shorter life span than nondiabetics, studies have been conducted on reducing platelet activity of diabetics with fish oil.
Vclardo et al., in Thromb. Haemostas 48 (3) 344 (1982) disclose that platelet activity in diabetics can be decreased by administration of high quantities of fish oil or eicosapentaenoic acid.
Haines et al., in Thromb. Res. 43: 643-655, 1986, disclose that a fish oil supplement was effective in reducing thromboxane production by platelets stimulated by collagen in diabetics. The fish oil supplements also increased plasma LDL cholesterol, fibrinogen, and clotting factor X in the group who took the fish oil supplement.
Neither of the two references above discloses any change in the blood glucose levels of the diabetics receiving fish oil.
The active ingredients in fish oil are (all-Z)-5,8,11,14,17-eicosapentaenoic acid (hereinafter EPA) and 22:6 omega3-docosahexaenoic acid (hereinafter DHA). EPA and DHA are known to be precursors in the biosynthesis of the prostaglandin PGE.sub.3.
It is disclosed in British Patents Nos. 1,604,554 and 2,033,745 that EPA can be used to treat effectively, or to provide effective prophylaxis against, thrombo-embolic conditions such as myocardial infarctions, strokes, or deep vein thrombosis during surgical operations. These patents disclose the extraction of EPA from fish oil such as cod liver oil or menhaden oil. The EPA may be administered by replacing butter or ordinary margarine by a special margarine formulated so that in normal usage the recipient would receive the required amount of the EPA.
This process has not achieved widespread attention, despite the fact that it uses a natural substance which can readily be incorporated into the daily diet. One reason may be due to the difficulty of efficiently separating EPA from natural fish oils to obtain a pure product at reasonable cost. Another reason may be that the effects of administration of EPA are not as dramatic as anticipated.
Prostaglandins are a family of substances showing a wide diversity of biological effects. Prostaglandins of the 1-, 2-, and 3-series, respectively, incorporate one, two, or three double bonds in their basic 20-carbon carboxylic fatty acid structure which incorporates a 5-member cyclopentene ring.
The 1-series of prostaglandins are strong vasodilators, and inhibit cholesterol and collagen biosynthesis, as well as platelet aggregation. On the other hand, the 2-series prostaglandins are known to enhance platelet aggregation, cholesterol, and collagen biosynthesis, and also to enhance endothelial cell proliferation. The main effect of the 3-series prostaglandins, particularly PGE3, is the suppression of the 2-series prostaglandins.
The precursor of the 2-series prostaglandins is arachidonic acid ((All-Z-5,8,11,14-eicosatetraenoic acid). DHLA is the precursor for the 1-series prostaglandins, and, as indicated hereinabove, EPA and DHA are precursors for the 3-series prostaglandins.
It is believed that EPA and DHA are effective precursors for prostaglandin PGE.sub.3, which suppresses the 2-series prostaglandins. Additionally, EPA and/or DHA itself competes with arachidonic acid on the same enzymatic system and thus inhibits the biosynthesis of 2-series prostaglandins. This inhibition of the 2-series prostaglandins results in an increase of the ratio of PGE.sub.1 :PGE.sub.2.